Genetic Testing for Cancer: Diagnostic Medicine & Cancer Susceptibility Gail H. Vance, M.D. Professor, Medical & Molecular Genetics Indiana University School of Medicine

Objectives  Differential genetic testing for somatic mutations from genetic testing for heritable mutations.  Use breast cancer as an example of disease utilizing both.  Demonstrate that both forms of testing provide information regarding risk and treatment

What is Cancer?

Cancer is a heterogeneous disease that will claim more than 560,000 lives in our country this year.

2009 Estimated US Cancer Deaths* ONS=Other nervous system. Source: American Cancer Society, Men 292,540 Women 269,800  26% Lung and bronchus  15%Breast  9%Colon and rectum  6%Pancreas  5%Ovary  3%Leukemia  3%Non-Hodgkin lymphoma  3%Uterine corpus  2%Liver  2%Brain/ONS  22% All other sites Lung and bronchus30% Prostate9% Colon and rectum9% Pancreas6% Leukemia4% Esophagus4% Liver and intrahepatic4% bile duct Non-Hodgkin 3% Lymphoma Urinary bladder3% Kidney3% All other sites 24%

Cancer is not a single disease.  Rather, any cancer may involve: Multiple tissue lineages. Aberrant expression of genes with a variety of cellular functions. Variation in the number of deregulated genes

Cancer is a genetic disease.  All cancers involve genetic changes in somatic cells, the germ line, or both.

What is Genetic Testing  The analysis of human DNA in any of its forms or related products (chromosomes, RNA, proteins)

Cancer Genes  Most gene mutations in cancer occur in somatic cells and are acquired (multifactorial etiology).  However, some mutations do occur in the germline and may be inherited and passed on to future generations.

 Individuals genetically predisposed to cancer account for 5-10% of the cancer population.  1,000,000 x 0.1=100,000 CANCER BURDEN

Features suggesting an inherited predisposition to cancer:  Two or more close relatives affected.  Early age of onset.  Cancers of a specific type occurring together (breast and ovary).  Multiple or bilateral cancers occurring in one person.

Breast Cancer  Breast cancer is a common disorder  Lifetime risk is 1 in 8.  180,000 new case each year

Breast Cancer  Two major susceptibility genes, BRCA1 and BRCA2 have been identified.  Mutations in these genes account for 3-5% of all breast cancers.

BRCA1 Gene on chromosome 17 Autosomal dominant transmission Protein has role in genomic stability >1300 different mutations reported

Gene on chromosome 13 Autosomal dominant transmission Protein has role in genomic stability >1300 different mutations reported BRCA2

BRCA1 and BRCA2-Linked Mutations in the Ashkenazi Jewish Population

Cancer Detection/Screening  Increased surveillance Monthly self-breast exams Semiannual clinical breast exams Semiannual radiography (mammogram/MRI) Annual transvaginal ultrasound, CA125

Cancer Prevention  Chemoprevention Tamoxifen Aromatase-inhibitors Oral contraceptives

Cancer Prevention  Prophylactic surgery Prophylactic mastectomy reduces risk by 90% Prophylactic salpingo-oophorectomy reduces risk by 96%

d. 53 BR CA 87 BR CA dx 80 d. 65 BR CA dx 48 d. 43 panc Ca 55 melanoma dx 49 d. 80d BR CA dx 58 85d d. 75d. 80 BRCA2 mutation Paternal lineage

Genetic Testing for Somatic Mutations in Breast Cancer  HER2 amplification  Oncotype Dx  MammaPrint

How do we assess risk in BC patients?  Classical pathological criteria  Lymph node  Age/Tumor size/Tumor grade  ER/PR/HER2  Oncotype Dx  MammaPrint

HER2 in Breast Cancer  Human Epidermal Growth Factor Receptor 2, HER2 (ERBB2) Amplified in ~20% breast cancers Prognostic marker Predictive for several systemic therapies  Relative resistance to endocrine therapies  Resistance or sensitivity to various types of chemotherapy  Response to agents that target HER2 (Herceptin ® )

Methods for HER2 Detection (IHC and FISH) AmplifiedNormal Red – HER2; Green – Control Probe Amplified: HER2/CEP17 > 2.2 or average HER2 > 6 Courtesy of D. Persons

Assessing HER2 with IHC Herceptin Treatment Indicated for 3+ and some 2+

Oncotype Dx  Molecular test performed on formalin-fixed breast tumors.  Used to assess recurrence risk and predicts benefit from hormonal/chemotherapy.  Utility for ER+, LN negative tumors.

Oncotype DX ® 21-Gene Recurrence Score ® (RS) Assay PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 ESTROGEN ER PR Bcl2 SCUBE2 INVASION Stromelysin 3 Cathepsin L2 HER2 GRB7 HER2 BAG1GSTM1 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC CD68 16 Cancer and 5 Reference Genes From 3 Studies Paik et al. N Engl J Med. 2004;351:

Oncotype DX ® 21-Gene Recurrence Score ® (RS) Assay Calculation of the Recurrence Score Result CategoryRS (0-100) Low riskRS <18 Int riskRS ≥18 and <31 High riskRS ≥31 Paik et al. N Engl J Med. 2004;351: RS = Coefficient x Expression Level x HER2 Group Score x ER Group Score x Proliferation Group Score x Invasion Group Score x CD x GSTM x BAG1 Coefficient x Expression Level x HER2 Group Score x ER Group Score x Proliferation Group Score x Invasion Group Score x CD x GSTM x BAG1 Coefficient x Expression Level RS= x HER2 Group Score x ER Group Score x Proliferation Group Score x Invasion Group Score x CD x GSTM X BAG1

Paik et al. N Engl J Med. 2004;351: The Recurrence Score ® Result Stratifies Patients by their 10-Year Distant Recurrence-Free Survival

MammaPrint  DNA microarray-based in vitro diagnostic test used to assess risk of cancer recurrence.  70 gene signature of critical molecular pathways associated with breast cancer metastasis.  Estimates high or low risk of recurrence.  Fresh frozen tumor specimens.

Summary  Genetic testing performed for both heritable and somatic gene mutations.  Heritable (germline) mutations may be predictive and performed in healthy women from blood.  Heritable mutations also predict risk for future disease in multiple organs.  Somatic mutation testing performed on the tumor to better assess risk and inform therapy.